Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2
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Updated: Jul 7, 2026
Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry
Published on: June 6, 2014
Peng Ji1, Senthil Raja Jayapal, Harvey F Lodish
1Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Floor 6, Cambridge, MA 02142, USA.
Abstract:
Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts.
Insights
Rac GTPases and their effector mDia2 are crucial for mammalian erythroblast enucleation, a process where the nucleus is expelled. Disrupting these proteins blocks the contractile actin ring formation, halting red blood cell maturation.
Area of Science:
- Cell Biology
- Hematopoiesis
- Molecular Mechanisms
Background:
- Erythroid cell enucleation is essential for red blood cell formation but its regulatory mechanisms are poorly understood.
- This process involves the asymmetric division and expulsion of the nucleus from developing red blood cells.
Purpose of the Study:
- To elucidate the molecular mechanisms regulating mammalian erythroblast enucleation.
- To investigate the role of Rac GTPases and their downstream effectors in this process.
Main Methods:
- Utilized cultured mouse fetal erythroblasts.
- Employed Rac GTPase inhibition and small interfering RNA (siRNA) to downregulate mDia2.
- Assessed enucleation, proliferation, differentiation, and contractile actin ring (CAR) formation.
- Investigated protein-protein interactions using GTP-dependent binding assays.
- Examined the effects of constitutively active mDia2 mutants.
Main Results:
- Deregulation of Rac GTPase completely inhibited erythroblast enucleation without affecting proliferation or differentiation.
- Inhibition of Rac GTPases disrupted the formation of the contractile actin ring (CAR).
- mDia2, a formin protein, was found to be essential for enucleation.
- Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner.
- Downregulation of mDia2 using siRNA blocked CAR formation and enucleation.
- Overexpression of active mDia2 rescued Rac GTPase inhibition-induced enucleation defects.
Conclusions:
- Rac GTPases play a critical role in regulating mammalian erythroblast enucleation.
- The formin protein mDia2 acts as a key downstream effector of Rac GTPases in this process.
- Rac GTPases and mDia2 are essential for the formation of the contractile actin ring during enucleation.
